Volume 12 Supplement 1

Breast Cancer Research 2010

Open Access

Transcriptional regulation of cyclin-dependent kinase inhibitor 1A (P21) by the transcription factor AP-2γ

  • AG Scibetta1,
  • M Canosa1 and
  • HC Hurst1
Breast Cancer Research201012(Suppl 1):P20

https://doi.org/10.1186/bcr2517

Published: 18 May 2010

Introduction

AP-2 transcription factors constitute a family of sequence-specific DNA-binding proteins encoded by five highly homologous yet functionally distinct genes, AP-2α to AP-2ε. AP-2γ appears to play a major role in breast cancer, being expressed in a large proportion of primary tumours. In this study we have analysed in more detail the mechanism of transcriptional regulation of the p21/cyclin-dependent kinase inhibitor 1A (p21/CDKN1A) gene by AP-2γ.

Materials and methods

Silencing of AP-2γ was carried out in MCF-7 cells using siRNA or doxycycline inducible shRNA. Chromatin immunoprecipitation (ChIP) assays were performed using specific antibodies against AP-2γ (H77), AP-2α, Myc, histone demethylase PLU1/JARID1B, histone H3 and trimethyl dimethyl and monomethyl histone H3 followed by quantitative PCR. Electrophoretic mobility shift assay (EMSA) competition assay and reporter assays were used to identify the AP-2 binding site.

Results

Silencing of AP-2γ by either siRNA or inducible shRNA inhibits cell proliferation and results in upregulation of p21/CDKN1A expression with no induction of apoptosis. ChIP assays demonstrated binding of AP-2γ, PLU1/JARID1B and Myc to a region adjacent to the transcription start site of the p21/CDKN1A gene. Reduction in the binding of cMyc and PLU1/JARID1B and increased levels of histone H3 trimethyl-K4 were observed at the proximal region of p21/CDKN1A promoter after silencing of AP-2γ. Treatment of MCF-7 cells with the antimitotic drug vinblastine but not with hydroxyurea reduced the CDKN1A binding of AP-2γ, PLU-1/JARID1B and Myc. H3396 cells treated with the oestrogen receptor inhibitor Faslodex, which upregulates p21/CDKN1A, decreased AP-2γ binding but increased binding of AP-2α at the p21/CDKN1A promoter. EMSA competition assays and reporter assays showed that AP-2γ and AP-2α bind to a new site (GCC N3 GGG) at position -105 of the p21/CDKN1A promoter.

Conclusions

The repression of the p21/CDKN1A gene by AP-2γ may contribute to the activation of proliferation associated with this transcription factor in breast cancer.

Authors’ Affiliations

(1)
Centre for Tumour Biology, Institute of Cancer, Queen Mary University of London

Copyright

© BioMed Central Ltd. 2010

Advertisement